Recombinant Pseudomonas syringae pv. syringae Trigger factor (tig)

What is the role of Trigger factor (tig) in Pseudomonas syringae pv. syringae?

Trigger factor (tig) in Pseudomonas syringae functions as a molecular chaperone that associates with ribosomes and facilitates co-translational protein folding. Based on comparative studies with other bacterial systems, tig likely plays a critical role in ensuring proper folding of virulence factors and other proteins essential for P. syringae pathogenicity. In P. syringae, tig may contribute to the proper folding of secreted effector proteins, particularly those involved in the type III secretion system (T3SS) that are critical for overcoming plant immune defenses . Functional analysis of tig in related Pseudomonas species suggests its involvement in stress responses, particularly during host infection processes when rapid protein synthesis and folding are required.

How does Trigger factor relate to P. syringae virulence mechanisms?

Trigger factor potentially contributes to P. syringae virulence by ensuring proper folding of key virulence-associated proteins. P. syringae employs several virulence strategies, including secreting effector proteins through the needle-like type III secretion system (T3SS), changing motility patterns, and producing phytotoxins . As a co-translational chaperone, tig likely ensures these proteins achieve proper conformation for functionality. Research indicates that P. syringae virulence is regulated by a hierarchical transcriptional network, with virulence-involved transcription factors categorized into three tiers . Tig may work in concert with this regulatory network by facilitating the proper folding of the transcription factors involved in virulence regulation.

What are the genomic characteristics of the tig gene in P. syringae pv. syringae?

The tig gene in P. syringae pv. syringae is part of the core genome conserved across Pseudomonas species. Based on genomic analyses of P. syringae strains, tig likely exhibits high conservation with orthologs in related bacterial species. Comparative genomic studies across P. syringae pathovars (including Psph 1448A, Pst DC3000, Pss B728a, and Psa C48) have revealed functional variability and different conservation patterns among various genes . While tig itself is highly conserved in its coding sequence, its regulatory elements may show strain-specific variations, potentially leading to differences in expression patterns across P. syringae pathovars.

What are the optimal expression systems for recombinant P. syringae Trigger factor?

The optimal expression system for recombinant P. syringae Trigger factor depends on research objectives and downstream applications. Two primary approaches are commonly employed:

Table 1: Comparison of Expression Systems for Recombinant P. syringae Trigger factor

For most biochemical and structural studies, E. coli-based expression is recommended due to higher yields and simpler purification. When using CHO cells for expression, consider using dihydrofolate reductase (DHFR) deficient CHO cell lines, as these allow for gene copy number amplification to potentially increase productivity . The efficiency of transgene expression is highly dependent on both the number of gene copies integrated and the location of integration in the genome .

What cloning strategies improve the expression of recombinant P. syringae Trigger factor?

Several cloning strategies can enhance the expression of recombinant P. syringae Trigger factor:

• Codon optimization: Adapt the P. syringae tig gene codons to match the codon usage bias of the expression host to improve translation efficiency.

• Fusion tags selection: For bacterial expression, consider:

  • N-terminal His6 tag for IMAC purification

  • MBP fusion for enhanced solubility

  • SUMO fusion for native N-terminus after protease cleavage

• Vector engineering: Advanced vector systems that incorporate:

  • Strong, inducible promoters (T7 for bacterial systems)

  • Optimal ribosome binding sites

  • Transcription terminators

• Site-specific integration: For CHO cell expression, site-specific integration techniques have shown promise for cell line development . The integration site significantly impacts the production of the gene of interest (GOI), with random integration of transgenes not always yielding consistent productivity despite high copy numbers .

• Genome editing: Consider CRISPR/Cas9, ZFNs, or TALENs systems for targeted integration of the tig gene in CHO cell lines, which can improve productivity while enhancing product quality .

How can I troubleshoot poor yield of recombinant P. syringae Trigger factor?

When encountering poor yield of recombinant P. syringae Trigger factor, consider the following systematic troubleshooting approach:

Table 2: Troubleshooting Guide for Recombinant Trigger Factor Expression

To improve protein production in CHO cells specifically, it's crucial to identify bottlenecks in transcription, translation, post-translational modifications, protein folding, and secretion . Cell engineering technology can precisely control the relative expression of multiple functional gene components, directly affecting protein yield. For challenging proteins, optimization should begin from molecular design, combined with appropriate cell line selection, vector engineering, and process optimization .

How can I study the interaction between P. syringae Trigger factor and transcription factors?

To study interactions between P. syringae Trigger factor and transcription factors, employ these methodological approaches:

• Co-immunoprecipitation (Co-IP): Express epitope-tagged tig and the transcription factor of interest, then perform pull-down assays followed by western blotting or mass spectrometry.

• Chromatin Immunoprecipitation (ChIP) for indirect interactions: If Trigger factor indirectly affects transcription factor activity, ChIP-seq can be employed to examine how tig affects transcription factor binding to DNA. Similar approaches have been used to study the binding characteristics of 170 transcription factors in P. syringae through chromatin immunoprecipitation sequencing .

• Yeast two-hybrid or bacterial two-hybrid assays: These systems can detect direct protein-protein interactions between tig and transcription factors.

• Surface Plasmon Resonance (SPR): Quantify binding kinetics and affinity between purified recombinant tig and transcription factors.

• Microscale Thermophoresis (MST): Measure interactions in solution with minimal protein consumption.

In P. syringae, particular attention should be given to transcription factors involved in virulence regulation. Research has identified multiple regulatory channels governing virulence in P. syringae, with transcription factors categorized into three tiers based on their regulatory position . Investigating how tig interacts with these different tiers of transcription factors could provide insights into its role in virulence regulation.

What methods can determine the structural features of P. syringae Trigger factor?

To elucidate the structural features of P. syringae Trigger factor, employ these complementary approaches:

For functional interpretation of structural data, correlate the identified domains with interactions with specific transcription factors or other cellular components. The structure-function relationship might provide insights into how tig contributes to the complex transcriptional regulatory network observed in P. syringae .

How can CRISPR-Cas9 be used to study Trigger factor function in P. syringae?

CRISPR-Cas9 offers powerful approaches for investigating Trigger factor function in P. syringae:

• Gene knockout studies:

  • Design sgRNAs targeting the tig gene

  • Introduce CRISPR-Cas9 components via electroporation

  • Screen transformants for successful editing

  • Assess phenotypic changes in growth, stress response, and virulence

• Domain-specific mutations:

  • Create point mutations in specific functional domains using CRISPR base editors

  • Analyze how specific mutations affect protein interactions and function

• Transcriptional regulation studies:

  • Use CRISPRi (CRISPR interference) with catalytically inactive Cas9 (dCas9) to modulate tig expression

  • Assess effects on downstream gene expression and phenotypes

• Epitope tagging:

  • Use CRISPR-Cas9 to introduce tags at the genomic locus for in situ studies

  • Allow visualization and purification of tig under native expression conditions

CRISPR-Cas9 systems, along with ZFNs and TALENs, have been successfully used to delete or introduce proteins in various cell systems while improving recombinant protein productivity . When applying these techniques to P. syringae, it's important to optimize transformation protocols and selection strategies for this specific bacterial system.

How does temperature affect the chaperone activity of P. syringae Trigger factor?

Temperature significantly impacts the chaperone activity of P. syringae Trigger factor, which is particularly relevant given the environmental adaptation needs of this plant pathogen:

• Temperature-dependent structural changes: Trigger factor undergoes conformational changes at different temperatures, affecting substrate binding affinity. At lower temperatures (15-20°C), which are common in the plant environment, P. syringae Trigger factor likely maintains higher flexibility in its substrate-binding domain.

• Cold adaptation mechanisms: Being a plant pathogen that often infects plants in cooler conditions, P. syringae Trigger factor may possess unique cold-adapted features compared to homologs from mesophilic bacteria.

• Experimental approach to measure temperature effects:

  • Express and purify recombinant P. syringae Trigger factor

  • Perform thermal shift assays to determine stability at different temperatures

  • Conduct substrate protection assays at temperature ranges relevant to plant infection (4-30°C)

  • Use circular dichroism spectroscopy to monitor secondary structure changes

• Physiological relevance: Temperature-dependent chaperone activity may contribute to P. syringae's ability to cause disease under various environmental conditions, particularly as temperature fluctuations can affect the expression of virulence factors .

When designing experiments to assess temperature effects, consider including other P. syringae proteins involved in virulence as potential substrates, as this may reveal how Trigger factor contributes to temperature-dependent pathogenicity.

What techniques can assess the impact of Trigger factor on P. syringae virulence gene expression?

To examine how Trigger factor influences P. syringae virulence gene expression, consider these methodological approaches:

• RNA-seq analysis:

  • Compare transcriptomes of wild-type and tig knockout/knockdown strains

  • Focus on differential expression of known virulence genes, particularly those in the T3SS pathway

  • Analyze data in context of the hierarchical transcriptional regulatory network described for P. syringae

• qRT-PCR validation:

  • Validate RNA-seq findings for key virulence genes

  • Measure expression of master regulators such as HrpL, HrpS, and RhpRS that control T3SS

• Reporter gene assays:

  • Construct promoter-reporter fusions for key virulence genes

  • Compare reporter activity in wild-type and tig-modified strains

• Chromatin immunoprecipitation sequencing (ChIP-seq):

  • If tig affects transcription factor activity, conduct ChIP-seq to map transcription factor binding sites

  • Compare binding profiles in presence/absence of functional tig

  • Similar approaches have been used to investigate binding characteristics of 170 transcription factors in P. syringae

• Plant infection assays:

  • Assess virulence phenotypes of tig mutants on host plants

  • Quantify bacterial growth in planta

  • Measure expression of virulence genes during infection

When interpreting results, consider that P. syringae uses hundreds of transcription factors to regulate functional processes, including virulence and metabolic pathways . Trigger factor may affect multiple regulatory levels within this complex network.

How can isothermal titration calorimetry be used to study P. syringae Trigger factor interactions?

Isothermal Titration Calorimetry (ITC) provides valuable thermodynamic data for studying P. syringae Trigger factor interactions with substrates and binding partners:

• Experimental design for ITC analysis:

  • Purify recombinant P. syringae Trigger factor to >95% homogeneity

  • Prepare potential binding partners (unfolded proteins, ribosomes, transcription factors)

  • Titrate binding partners into tig solution under controlled temperature

  • Record heat changes to determine binding parameters

• Thermodynamic parameters obtainable:

  • Binding affinity (Kd)

  • Enthalpy change (ΔH)

  • Entropy change (ΔS)

  • Stoichiometry (n)

  • Gibbs free energy change (ΔG)

• Specific applications for P. syringae research:

  • Measure binding affinity between tig and nascent polypeptides

  • Characterize interactions with specific transcription factors implicated in virulence

  • Study temperature-dependence of binding (relevant for plant infection)

  • Determine effects of mutations on binding properties

• Data interpretation guidelines:

  • Exothermic reactions (negative ΔH) typically indicate hydrogen bonding and van der Waals interactions

  • Endothermic reactions (positive ΔH) often reflect hydrophobic interactions

  • Changes in binding parameters under different conditions can reveal mechanisms of regulation

For P. syringae specifically, ITC could help determine if Trigger factor preferentially binds to virulence-associated proteins compared to housekeeping proteins, potentially explaining its role in pathogenicity.

How does P. syringae Trigger factor compare to homologs in other bacterial species?

P. syringae Trigger factor exhibits both conserved features and species-specific adaptations when compared to homologs in other bacteria:

Table 3: Comparative Analysis of Bacterial Trigger Factors

Evolutionary analysis of P. syringae strains reveals both functional variability and conservation patterns among various genes . While core chaperone functions of Trigger factor are likely conserved across bacteria, substrate specificity and regulatory mechanisms may have evolved to support P. syringae's plant pathogenic lifestyle. Comparative analysis of binding motifs across P. syringae strains (Psph 1448A, Pst DC3000, Pss B728a, and Psa C48) has revealed similar core binding sequences but distribution in different target genes, suggesting functional diversification .

What evolutionary insights can be gained from studying P. syringae Trigger factor?

Studying P. syringae Trigger factor from an evolutionary perspective can provide several valuable insights:

• Adaptation to plant host environments:

  • Analyze selection pressures on different Trigger factor domains

  • Compare sequences across P. syringae pathovars that infect different plant hosts

  • Identify positively selected residues that may contribute to host-specific adaptation

• Co-evolution with virulence mechanisms:

  • Examine how Trigger factor evolution correlates with evolution of T3SS components

  • Investigate potential co-evolution with transcription factors that regulate virulence

• Genomic context conservation:

  • Analyze conservation of tig gene location and flanking regions

  • Compare operon structures across Pseudomonas species

• Methodological approach:

  • Perform phylogenetic analysis of tig sequences from diverse P. syringae strains

  • Calculate dN/dS ratios to identify regions under positive or purifying selection

  • Use ancestral sequence reconstruction to infer evolutionary trajectory

What are the best approaches for studying Trigger factor-ribosome interactions in P. syringae?

To effectively study Trigger factor-ribosome interactions in P. syringae, consider these methodological approaches:

• Cryo-electron microscopy (Cryo-EM):

  • Purify P. syringae ribosomes with associated Trigger factor

  • Collect high-resolution images of the complex

  • Perform 3D reconstruction to visualize binding interface

  • Identify specific ribosomal proteins involved in the interaction

• Ribosome profiling with Trigger factor mutants:

  • Generate tig knockout or ribosome-binding deficient mutants

  • Perform ribosome profiling to map ribosome positioning on mRNAs

  • Analyze changes in translation efficiency, particularly for virulence factors

• Fluorescence-based interaction assays:

  • Label Trigger factor and ribosomal proteins with fluorescent tags

  • Measure binding kinetics using fluorescence resonance energy transfer (FRET)

  • Determine how environmental factors affect the interaction

• Chemical cross-linking coupled with mass spectrometry:

  • Cross-link Trigger factor to ribosomes in vivo or in vitro

  • Digest complexes and identify cross-linked peptides by mass spectrometry

  • Map the interaction interface at amino acid resolution

• Co-sedimentation assays:

  • Mix purified Trigger factor with isolated ribosomes

  • Perform ultracentrifugation through sucrose cushions

  • Analyze co-sedimentation to quantify binding affinity

  • Test effects of buffer conditions, temperature, and mutations

When interpreting results, consider the hierarchical information flow in P. syringae regulatory networks . Ribosome-Trigger factor interactions may vary depending on the nature of the nascent chain, with potential preferences for proteins involved in virulence or stress response.

How can I develop an activity assay for P. syringae Trigger factor?

Developing robust activity assays for P. syringae Trigger factor requires careful consideration of its dual peptidyl-prolyl isomerase (PPIase) and chaperone functions:

• PPIase activity assay:

  • Utilize synthetic tetrapeptides containing proline (e.g., Ala-Ala-Pro-Phe-pNA)

  • Monitor spectrophotometric changes upon cis-trans isomerization

  • Measure reaction rates at different temperatures (5-30°C) to reflect P. syringae's environmental range

  • Calculate kinetic parameters (kcat, KM) under various conditions

• Chaperone activity assay:

  • Monitor prevention of aggregation of model substrates (e.g., citrate synthase, rhodanese)

  • Use light scattering at 320-360 nm to quantify aggregation suppression

  • Include P. syringae-specific virulence proteins as substrates

  • Test activity under conditions mimicking plant infection

• Ribosome-bound activity assessment:

  • Purify P. syringae ribosomes with associated nascent chains

  • Compare folding of nascent polypeptides with and without added Trigger factor

  • Monitor by limited proteolysis or conformation-specific antibodies

• Fluorescence-based folding assays:

  • Engineer fluorescent protein fusions that report on folding state

  • Measure folding kinetics in presence and absence of Trigger factor

  • Quantify through changes in fluorescence properties

• In vivo complementation assay:

  • Express P. syringae Trigger factor in E. coli tig deletion strain

  • Assess restoration of growth at low temperatures or under stress

  • Compare with native E. coli Trigger factor to identify functional differences

When developing these assays, consider that P. syringae Trigger factor may have evolved specialized functions related to virulence factor folding, potentially reflected in substrate preferences or temperature optima different from model organisms.

What cell-free expression systems are suitable for producing recombinant P. syringae Trigger factor?

Cell-free expression systems offer distinct advantages for producing recombinant P. syringae Trigger factor, particularly for difficult-to-express variants or when rapid production is needed:

Table 4: Comparison of Cell-Free Expression Systems for P. syringae Trigger Factor

Methodological considerations for cell-free expression:

• Template preparation:

  • Optimize codon usage for the cell-free system

  • Include appropriate regulatory elements (T7 promoter, enhancers)

  • Consider PCR-based template generation for rapid screening

• Reaction optimization:

  • Adjust magnesium and potassium concentrations for maximum yield

  • Supplement with molecular chaperones for improved folding

  • Implement continuous exchange systems for longer reaction times and higher yields

• Co-translational modifications:

  • Add microsomes or nanodiscs for membrane-associated studies

  • Include specific tRNAs for site-specific modification or unnatural amino acid incorporation

• Scale-up strategies:

  • Implement microfluidic or dialysis systems for larger-scale production

  • Optimize energy regeneration systems for sustained expression

Cell-free systems are particularly valuable for studying the function of Trigger factor itself, as they allow direct observation of its co-translational activity without interference from endogenous tig in the expression host.

How does Trigger factor interact with the transcriptional regulatory network in P. syringae?

The interaction between Trigger factor and the transcriptional regulatory network in P. syringae likely involves multiple mechanisms:

• Co-translational folding of transcription factors:

  • Trigger factor may preferentially assist the folding of key transcription factors

  • Research has identified 54 top-level, 62 middle-level, and 147 bottom-level transcription factors in P. syringae, forming a hierarchical regulatory network

  • The proper folding of these transcription factors is critical for their regulatory function

• Influence on regulatory cascades:

  • P. syringae virulence is controlled by transcriptional regulatory channels organized in a three-tier hierarchy

  • Trigger factor may differentially affect the stability and activity of transcription factors at different levels of this hierarchy

  • This could create regulatory patterns where tig availability influences entire virulence pathways

• Experimental approaches to study these interactions:

  • Compare the proteome stability of wild-type and tig-deficient strains

  • Conduct ChIP-seq in tig mutant backgrounds to assess changes in transcription factor binding

  • Perform quantitative proteomics to identify transcription factors whose levels are tig-dependent

• Potential regulatory feedback:

  • Transcription factors may in turn regulate tig expression under specific conditions

  • This creates potential for complex feedback loops in the regulatory network

The complex transcriptional regulatory network in P. syringae, with its multiple higher-order network structures and directional information flow , provides numerous points where Trigger factor could influence gene expression through its effect on protein folding and stability.

What is the relationship between Trigger factor and other chaperones in P. syringae?

Trigger factor operates within a complex chaperone network in P. syringae, with functional relationships and potential redundancies:

• Cooperative chaperone systems:

  • Trigger factor likely works in concert with the DnaK/DnaJ/GrpE system and GroEL/GroES chaperonins

  • These systems form a sequential folding pathway for newly synthesized proteins

  • In E. coli, deletion of both tig and dnaK is synthetically lethal; similar relationships likely exist in P. syringae

• Specialized roles in P. syringae pathogenicity:

  • Trigger factor may specialize in folding secreted virulence factors

  • Other chaperones might focus on cytoplasmic proteins or stress responses

  • The distribution of substrates between different chaperone systems may be optimized for pathogenicity

• Experimental approaches to map chaperone networks:

  • Generate single and double chaperone mutants to identify genetic interactions

  • Perform comparative substrate profiling using pull-down assays

  • Use proteomics to identify proteins whose stability depends on specific chaperones

• Regulatory coordination:

  • Examine whether chaperones are co-regulated during infection or stress

  • Investigate if the hierarchical transcriptional regulatory network in P. syringae coordinates chaperone expression

  • Study how environmental conditions affect the relative importance of different chaperones

Understanding this chaperone network is particularly relevant given P. syringae's need to adapt to changing environmental conditions during plant infection, where temperature fluctuations and host defenses create varying proteostasis challenges.

How can recombinant P. syringae Trigger factor be used to study plant immune responses?

Recombinant P. syringae Trigger factor offers several experimental approaches for investigating plant immune responses:

• Plant receptor recognition studies:

  • Test if plant pattern recognition receptors detect Trigger factor as a microbe-associated molecular pattern (MAMP)

  • Measure immune responses (ROS burst, MAP kinase activation, defense gene expression) after treatment with purified tig

  • Compare responses in different plant species and immune receptor mutants

• Interaction with plant defense signaling:

  • Investigate if Trigger factor interacts with or modifies plant defense proteins

  • Assess if tig affects the stability of plant immune components during infection

  • Examine potential interference with plant protein folding machinery

• Comparative immunity assays:

  • Compare plant responses to wild-type P. syringae versus tig mutants

  • Quantify differences in bacterial growth, symptom development, and defense gene activation

  • Determine if tig contributes to evasion of plant immunity

• Vaccination potential:

  • Evaluate if pre-treatment with recombinant tig induces systemic acquired resistance

  • Test protection against subsequent P. syringae infection

  • Identify plant signaling components required for any observed protection

This research direction connects to the broader understanding of P. syringae virulence mechanisms, where the bacterium deploys multiple strategies to overcome plant immune defenses . Trigger factor may play a previously unrecognized role in this process, either directly through interaction with plant components or indirectly by ensuring proper folding of bacterial virulence factors.

Can structural insights from P. syringae Trigger factor inform antimicrobial development?

Structural analysis of P. syringae Trigger factor can provide valuable insights for developing novel antimicrobials:

• Structure-based drug design approach:

  • Solve high-resolution structure of P. syringae Trigger factor, focusing on unique features

  • Identify druggable pockets, particularly those distinct from human chaperones

  • Perform in silico screening to identify potential inhibitors

  • Validate hits with biochemical assays and bacterial growth inhibition tests

• Targeting tig-specific functions in pathogenicity:

  • Design compounds that specifically inhibit interaction with virulence-related substrates

  • Develop molecules that block ribosome binding while preserving host protein function

  • Create peptide mimetics that compete with natural substrates

• Exploiting species-specific features:

  • Compare structures across bacterial species to identify Pseudomonas-specific regions

  • Target structural elements involved in adaptation to plant environment

  • Develop narrow-spectrum antimicrobials with reduced impact on beneficial microbes

• Rational design strategy:

  • Focus on compounds that destabilize virulence factors rather than killing bacteria directly

  • This approach may reduce selection pressure for resistance

  • Target bacterial fitness in planta rather than growth in laboratory conditions

This approach aligns with the growing understanding of P. syringae's complex virulence mechanisms and regulatory networks . By targeting a fundamental protein folding chaperone with specificity for key virulence factors, it may be possible to develop antimicrobials that reduce pathogenicity without strong selection for resistance.

What emerging technologies hold promise for studying P. syringae Trigger factor function?

Several cutting-edge technologies offer new opportunities for investigating P. syringae Trigger factor function:

• Single-molecule techniques:

  • Optical tweezers to study co-translational folding in real-time

  • Single-molecule FRET to monitor conformational changes during substrate binding

  • These approaches can provide unprecedented insights into the dynamics of tig action

• Advanced structural biology methods:

  • Cryo-electron tomography to visualize tig-ribosome complexes in situ

  • Integrative structural biology combining multiple data sources (X-ray, NMR, SAXS, cross-linking)

  • AlphaFold2 and other AI-based structure prediction tools to model tig-substrate complexes

• Spatial transcriptomics and proteomics:

  • Map tig localization and activity within bacterial cells during infection

  • Correlate with localization of transcription factors and virulence proteins

  • Create spatial maps of folding activity during host-pathogen interaction

• Synthetic biology approaches:

  • Engineer synthetic tig variants with altered substrate specificity

  • Create orthogonal tig-substrate pairs for probing specific pathways

  • Develop optogenetic or chemically inducible tig systems for temporal control

• Nanobody development:

  • Generate conformation-specific nanobodies against tig

  • Use as molecular probes for specific functional states

  • Apply in cellular imaging and functional perturbation studies

These technologies could help address fundamental questions about how Trigger factor contributes to P. syringae's complex transcriptional regulatory network and its role in coordinating virulence and metabolic pathways during infection.

What interdisciplinary approaches could advance P. syringae Trigger factor research?

Advancing P. syringae Trigger factor research can benefit from interdisciplinary approaches that integrate multiple scientific domains:

• Computational biology and molecular dynamics:

  • Simulate tig-substrate interactions at atomic resolution

  • Model folding trajectories of nascent polypeptides

  • Predict how mutations affect function based on molecular dynamics

• Systems biology integration:

  • Map tig function within the context of whole-cell models

  • Integrate with transcriptional regulatory networks in P. syringae

  • Model how perturbations in tig function propagate through cellular networks

• Evolutionary and ecological perspectives:

  • Compare tig function across P. syringae isolates from different hosts

  • Relate sequence variations to ecological adaptations

  • Study co-evolution with plant defense systems

• Agricultural biotechnology applications:

  • Develop tig-based strategies for crop protection

  • Engineer plants with enhanced recognition of tig or tig-dependent pathways

  • Create diagnostic tools based on tig conservation patterns

• Chemical biology approaches:

  • Develop activity-based probes for tig function

  • Screen for small molecules that modify tig activity

  • Engineer tig variants with novel functions or substrate specificities

These interdisciplinary approaches can provide a more comprehensive understanding of tig's role in P. syringae biology and pathogenicity, potentially identifying new targets for intervention in plant disease management.